Project Summary Diabetic nephropathy represents the primary cause of end stage renal disease (ESRD) in the US, underscoring the need for innovative therapies for preventing its progression. We are interested in understanding the cellular and molecular mechanisms that govern mitochondrial dysfunction in the diabetic milieu with the expectation that understanding of these processes will expose potential disease mechanisms and therapeutic targets in diabetic nephropathy. The present proposal is based on our recent published observation, indicating that mitochondrial fragmentation is essential for prompting mitochondrial dysfunction in podocytes in the diabetic milieu. A detailed understanding of mechanisms that govern mitochondrial fission in the kidney remains incomplete and therapeutic targets based on these mechanisms do not exist. Because dynamin-related protein-1 (Drp1) plays an integral part in regulating mitochondrial fission, we have focused on investigating the role of Drp1 in mitochondrial fragmentation and progression of diabetic nephropathy. We have been guided by our recent published observations that high glucose leads to mitochondrial fragmentation by promoting Drp1 recruitment to the mitochondria. Deletion of Drp1 in db/db diabetic mice prevented mitochondrial fission and improved histological and biochemical features of advanced diabetic kidney disease. Importantly, we found that high glucose milieu triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. Here, we propose to establish the crosstalk between phosphorylation of Drp1 and electron transport complexes (ETC) as key mediators of mitochondrial ROS (mROS) and potential therapeutic targets in diabetic nephropathy (DN). In support of our hypothesis, we have recently generated a novel diabetic knockin mutant mouse harboring a single phosphorylation deficient (serine-to-alanine) point mutation at the corresponding S600 site in the endogenous Drp1 allele (Drp1S600A). We observed that diabetic Drp1S600A mice exhibited improved key biochemical and histological features of DN. To assess the role of Drp1S600 phosphorylation on mROS, We next crossed diabetic Drp1S600A mice with mice that express a redox- sensitive green fluorescent protein biosensor (roGFP) specifically in the mitochondrial matrix (mt-roGFP) and observed that Drp1S600A mutation in diabetic mice leads to reduced mROS in podocytes in live diabetic animals. These findings provide compelling initial evidence into the unexpected role of Drp1 in a signaling cascade that regulates mROS, and represents a therapeutic target that might be useful in preventing diabetic kidney disease. Given these results and additional preliminary data presented in this application, this project will address the hypothesis that Drp1 phosphorylation dynamically interact with mitochondrial ETC to enhance mROS though a signaling network that is regulated by cardiolipin activation. The results of this study will provide important new insights into the role of mitochondrial morphology in the development of diabetic nephropathy, and may lead to novel therapeutic targets for the future treatment of diabetic kidney disease.